Denervation activated super-enhancers of pathogenic IL6-STAT3 feedforward loop in FAPs

项目总结 这项建议研究致病的IL6-STAT3的结构性激活的表观遗传学机制 来自失神经肌肉的纤维脂肪前体细胞(DEN FAP)的信号传导。FAP是必不可少的 骨骼肌细胞效应器对环境扰动的适应能力。为了应对急性损伤， FAP在炎症细胞和卫星细胞(SCs)的协调激活中被激活，并有助于 促进肌肉修复。相比之下，对慢性损伤(如肌肉营养不良)的反应是失调的 FAP、巨噬细胞(MPS)和SCs之间的功能相互作用导致再生受损和 因纤维化和脂肪沉积而导致的适应不良修复。我们最近发现了FAP的另一种模式 肌肉失神经后的激活，从而在没有MPS和MPS伴随的渗透的情况下 SC激活，DEN FAP的渐进扩展，IL6-STAT3信号的持续激活导致 肌纤维萎缩和纤维化。我们假设，基因组顺式调节元件的灵活使用能够 FAP采用不同的功能表型来响应不同类型的骨骼肌扰动， 通过激活特定的基因表达模式。在这方面，IL6的差异激活 FAP对不同类型扰动(即急性损伤与失神经)反应过程中的转录观察 提供了一个机会来研究顺式调控元件之间的动态相互作用如何调节 基因转录对不同刺激的反应阈值、幅度和持续时间。我们将调查 假设在DEN FAPs中，STAT3促进远端增强子和高度的 IL6转录起始点(TSS)附近的保守调控元件，激活组成性高 IL6转录和分泌水平，进而通过自分泌推动STAT3的长时间激活 IL-6受体的参与。由于FAP由动态中的离散子总体(子FAP)组成 在平衡条件下，我们假设IL6-STAT3信号的结构性激活导致了一种自我- 说明DEN FAP致病活性的自治的、占主导地位的亚群。具体的 AIMS将检验这样的假设，即STAT3介导的IL6超级增强子的激活是 建立致病前馈IL6-STAT3环。在目标1(DEN FAP中IL6基因的3D调控)中， 我们将使用染色体构象捕获(3C)和基于芯片的技术来识别和表征 DEN FAPs中的IL6基因相互作用增强子。目标2(检测DEN亚FAP及其和 功能特征)，我们将利用单细胞RNAseq来检测DEN亚FAP并建立 IL-6-STAT3过度激活与其自我扩张的功能关系在AIM 3中 在DEN FAP中，我们将使用STAT3激活的超级增强子的药理学靶向 药物抑制STAT3激活的超级增强子以确定是否阻断IL6- STAT3前馈环防止DEN亚FAP的致病潜力。

PROJECT SUMMARY This proposal investigates the epigenetic mechanism of constitutive activation of pathogenic IL6-STAT3 signaling in Fibro-Adipogenic Progenitors (FAPs) from denervated muscles (DEN FAPs). FAPs are essential cellular effectors of skeletal muscle ability to adapt to environmental perturbations. In response to acute injury, FAPs are activated within a coordinate activation of inflammatory and satellite cells (SCs), and contribute to promote muscle repair. By contrast, in response to chronic injury (e.g. muscular dystrophies) dysregulated functional interactions between FAPs, macrophages (MPs) and SCs lead to impaired regeneration and maladaptive repair by fibrosis and fat deposition. We have recently discovered an alternative pattern of FAPs activation in response to muscle denervation, whereby in the absence of concomitant infiltration of MPs and SC activation, a progressive expansion of DEN FAPs with persistent activation of IL6-STAT3 signaling leads to myofiber atrophy and fibrosis. We posit that a flexible usage of cis-regulatory elements of the genome enables FAPs to adopt different functional phenotypes in response to distinct types of skeletal muscle perturbations, through activation of specific patterns of gene expression. In this regard, the differential activation of IL6 transcription observed during FAP’s response to different types of perturbation (i.e. acute injury vs denervation) provides an opportunity to investigate how dynamic interactions between cis-regulatory elements regulate threshold, magnitude and duration of gene transcription in response to distinct stimuli. We will investigate the hypothesis that in DEN FAPs STAT3 promotes long-range interactions between distal enhancers and a highly conserved regulatory element proximal to the IL6 transcription start site (TSS), to activate constitutive high levels of IL6 transcription and secretion, which in turn drives prolonged activation of STAT3, via autocrine engagement of IL6 receptor. As FAPs are composed of discrete sub-populations (subFAPs) in dynamic equilibrium, we hypothesize the constitutive activation of IL6-STAT3 signaling leads to the emergence of a self- autonomous, dominant subpopulation that accounts for the pathogenic activity of DEN FAPs. The specific Aims will test the hypothesis that STAT3-mediated activation of IL6 super-enhancers is a key event for the establishing a pathogenic feedforward IL6-STAT3 loop. In Aim 1 (3D regulation of IL6 locus in DEN FAPs), we will use chromosome conformation capture (3C)- and ChIP-based technologies to identify and characterize IL6 gene-interacting enhancers in DEN FAPs. In Aim 2 (Detection of DEN subFAPs and their and functional characterization), we will exploit single cell RNAseq to detect DEN subFAPs and establish a functional relationship between IL6-STAT3 hyperactivation and their self-autonomous expansion. In Aim 3 Pharmacological targeting of STAT3-activated super-enhancers in DEN FAPs, we will use pharmacological inhibition of STAT3-activated super-enhancers to determine whether interruption of IL6- STAT3 feedforward loop prevents the pathogenic potential of DEN subFAPs.